The major goals of Project 1 include discovery and biochemical characterization of key proteases in cancer growth, angiogenesis, and metastasis. Project 1 is the biochemistry arm of the PPG effort and, in conjunction with the Biochemistry Core and the Chemistry Core, is responsible for identifying critical protease activities in tumors, evaluating known protease inhibitors, and developing new inhibitors. Various technologies will be used to search for the natural substrates of key proteases. In conjunction with the Chemistry Core, protease substrate libraries will be screened and selective inhibitors will be developed for "chemical genetic" experiments. Chemical reagents will also be developed for selectively labeling the proteases in vivo. Due to the progress made in the previous granting period on the discovery of new epithelial cancer-associated proteases, emphasis will be placed on the serine proteases, MT-SP1 and MT-SP2 in this funding cycle. Animal models will be used to determine the importance of MT-SP1 in the development and progression of prostate cancer. A tissue-specific MT-SP1 knockout mouse will be made in conjunction with the Animal Core and the effect of the knockout on transgenic models of cancer will be determined. The TRAMP transgenic mouse model will be used to determine the importance of MT-SP1 in prostate cancer and provide additional evidence for anti-protease therapy in different stages of the development and progression of epithelial cancers. Furthermore, our successful demonstration of engineering macromolecular protease inhibitors from ecotin or single chain antibody scaffolds will be extended to develop additional selectivity and potency. These inhibitors will serve as functional probes of protease activity and will be used in conjunction with Project 2 to determine their effect on other cancer models for purposes of experimental therapeutics. The antibodies will also be used for the development of ELISAs for monitoring levels of these proteases in cancer specimens and patients. While this project focuses on MT-SP1 and 2, the technology can also be applied to other serine, cysteine and metalloproteases found to be exploitable targets in conjunction with the Biochemistry Core and Projects 2 and 3.